William O. Whetsell

Immunoreactive Epidermal Growth Factor Receptors in Neuritic Plaques from Patients with Alzheimer's Disease

Alzheimers disease (AD) is characterized neuropathologically by the presence of neuritic plaques (NP) in cerebral cortex and hippocampus, as well as intraneuronal neurofibrillary tangles and granulovacuolar degeneration. The etiology of plaque formation has remained obscure, but morphologically NPare known to contain amyloid cores surrounded by astrocytes and degenerating neurons. Although growth factors are important in growth, differentiation and regrowth in response to injury, studies relating growth factors to ADhave been lacking. Epidermal growth factor (EGF) plays an important role outside the central nervous system (CNS) through interaction with its specific receptor, EGF-R. Using an antibody to EGF-R(threestep immunoperoxidase staining) in conjunction with fluorescence staining, we found that the majority of NPfrom patients with pathologically confirmed AD as well as those few NPin the normal aging brain showed intense EGF-Rimmunoreactivity. Specific staining was seen at the periphery of plaques but not in the central amyloid core. Tissue sections from ADcases were also reacted with antibodies to both glial fibrillary acidic protein (GFAP) and paired helical filaments (PHF) in an attempt to identify which component of the NPwas reactive for EGF-R.The antibody to PHFdensely stained the periphery of NP but not the central core in a majority of NP. The antibody to GFAPstained a few reactive astrocytes that bordered plaques in only a small proportion of all plaques present. We conclude that the neuron and its processes although not exclusively may be the site of EGF-Rimmunoreactivity. An EGF/EGF-Rsystem within the CNS may play an important part in scar formation in response to neuronal injury and death or it may function as a trophic factor important in axonal or dendritic sprouting. It is also possible that EGFcould serve as a neurotransmitter/neuromodulator in the CNS

Studies on porphyrin-heme biosynthesis in organotypic cultures of chick dorsal root ganglion. I. Observations on neuronal and non-neuronal elements.

Living, mature organotypic cultures of chick dorsal root ganglion maintained in culture for 3 weeks were incubated in medium containing various levels of a precursor of porphyrin and heme formation, delta-aminolevulinic acid (ALA), (0.5 mM to 10 mM) or a combination of ALA (10 MM) and a metal chelator, CaMg-EDTA (5 mM) for up to 48 hours. Although no morphologic changes occurred in the cultures incubated with these compounds as observed by bright-field or dark-field microscopy, fluorescence microscopic study at 12, 24, and 48 hours demonstrated an intense red fluorescence with in the non-neuronal cells of the cultures (Schwann cells, fibroblasts, and macrophages) but not in the nerve cells. Spectrofluorometric analysis of perchloric acid-methanol extracts of the cultures revealed an emission spectrum characteristic of porphyrins. Autoradiographic studies, using 14C-labelled ALA, indicated that ALA was taken up by all cells (nerve cells as well as non-neuronal cells) in the cultures. The cultures incubated with ALA plus the metal chelator CaMg-EDTA showed the same distribution of porphyrin fluorescence, but a 2-fold increase in the amount of porphyrins was generated, when compared to cultures incubated with ALA alone. This observation suggests that a considerable fraction of porphyrins may be utilized to form heme in these cells since CaMa-EDTA blocks ferrochelatase activity, the terminal enzyme in the heme biosynthetic pathway. This is the first demonstration of active porphyrin-heme biosynthesis from ALA in cultured nervous system cells. Our results indicate that this biosynthetic pathway remains active in 3-week old cultures of chick dorsal root ganglion, and further, that the pathway appears to be predominantly present in the non-neuronal cellular elements of the ganglion rather than in nerve cells.

Latent herpes simplex virus infection of mice. Infectious virus in homogenates of latently infected dorsal root ganglia.

C-57 albino weanling mice were latently infected with herpes simplex virus (Mp strain, type 1) by inoculation of 104 plaque forming units in the right hind footpad. The virus was demonstrable in explant cultures of the sacral dorsal root ganglia of these mice for as long as 18 months following inoculation. In addition, the virus was detectable when homogenates of these latently infected ganglia were placed on to differentiated organotypic cultures of fetal mouse dorsal root ganglia for as long as 8 months following inoculation of the mice. Virus was not demonstrable in these homogenates when they were placed on to Hela cells. The results suggest that during herpes simplex virus latent infection in mice there is continuous synthesis of infectious virus, probably in a highly localized area, which is detectable if a sensitive indicator substrate, such as these organotypic cultures, is used.

Comparative effects of herpes simplex virus types 1 and 2 in organotypic cultures of mouse dorsal root ganglion.

Mature organized cultures of mouse dorsal root ganglion (MDRG) were infected with herpes simplex virus, type 1 (HSV 1) and type 2 (HSV 2). Onset of infectious virus production occurred faster and reached higher levels in HSV 2-infected cultures. Neurons, supporting cells and myelin were affected in both types of infection, but morphological changes occurred significantly earlier and more dramatically with the type 2 infection. The pattern of myelin changes was distinctly different in the two types of infection. Within 20 hours post infection nerve cells infected with HSV 2 developed several types of intranuclear inclusions consisting of membranes and filaments; no such neuronal inclusions were seen with HSV 1 infection. HSV 2 infection showed frequent, large, membranous inclusions in supporting cell nuclei whereas, only rare, small inclusions of this type were seen in supporting cells infected with HSV 1. The observations demonstrate that the two virus types produce different virus replication pattern and different morphologic changes in long term cultures of MDRG. There appears to be a differential response of neurons and non-neuronal elements to the virus in the tissue substrate. Viral latency was not induced in this system by direct inoculation of the virus under the conditions described.

Herpes simplex virus types 1 and 2 in organotypic cultures of mouse central and peripheral nervous system. 2. Electron microscopic observations of myelin degeneration.

Organotypic cultures of mouse spinal cord with attached dorsal root ganglia, which contain both central and peripheral myelin in the one unit of tissue, were infected with HSV 1 or HSV 2 and studied using electron microscopy. Intranuclear viral nucleocapsids and intrcytoplasmic enveloped particles were found in the Schwann cells associated with peripheral myelin and in oligodendroglia associated with central myelin. Degeneration of peripheral myelin most commonly involved an asymmetrical swelling of the myelin lamellae, whereas degeneration of central myelin was characterized by a more generalized swelling resulting in separation of the myelin lamellae. Degeneration of both central and peripheral myelin was found in the presence of intact axons which were indistinguishable from those in controls.

Herpes Simplex Virus Types 1 and 2 in Organotypic Cultures of Mouse Central and Peripheral Nervous System

Mature mouse spinal cord-ganglion cultures, which contain both peripheral and central nervous system as one unit, were infected with herpes simplex virus type 1 (HSV 1) or type 2 (HSV 2) and observed by bright field microscopy for up to 72 hours. There was degeneration of both central and peripheral myelin in cultures infected with either virus, but the pattern of peripheral myelin degeneration associated with HSV 1-infected cultures was different from that in HSV 2-infected cultures. Type 1 was characterized by focal dilatations; type 2 by ‘sausage-shaped’ swellings, and the cytopathic effect of HSV 2 both began (6 hours p.i.) and was completed (36 hours p.i.) earlier than in cultures infected with HSV 1 (12 hours and 48 hours p.i. respectively). In central nervous tissue, the appearance of degenerating myelin after infection with HSV 1 was indistinguishable from that in HSV 2-infected cultures, but the rate of myelin loss was greater in cultures infected with the type 2 virus. Evidence is presented which suggests that, at least in the peripheral nervous system, myelin degeneration did not appear to be dependent on neuronal or axonal dysfunction or death, but was a direct result of virus infection.

Further Characterization of Brain Actin by Electron Microscopy

The physical state of actin in nerve ending preparations and its relationship to the membranes was studied at the ultrastructural level by negative staining with uranyl acetate before and after treatment with muscle heaving meromyosin (HMM). Actin prepared from synaptosomal or synaptic membrane preparations did not polymerize to fiber formation as readily as striated muscle actin under the same conditions. Treatment of these brain actin preparations with HMM, however, resulted in formation of fibers characteristically decorated with arrowheads which were quite similar to those formed with muscle actin. Treatment of the synaptosomal or synaptic membrane fractions themselves with HMM caused the formation of numerous decorated fibers although fibers were not evident before HMM treatment. This did not occur with the presynaptic vesicle fraction. The studies suggest that at least part of the actin is associated with synaptic membranes and is in a partially polymerized or non-polymerized state; polymerization can be induced by HMM.